Respiratory syncytial virus (RSV), a member of the paramyxovirus family, is the leading cause of infant hospitalization from infectious diseases in the United States. Regular re-infection of adults during seasonal epidemics provides the ground for caregiver-to-baby transmissions, which aggravate the problem. Despite extensive research, no vaccine protection is available and current antibody therapy-based immunoprophylaxis remains reserved for high-risk patients. Recognizing the unmet pediatric clinical need for efficacious, readily applicable and cost-effective RSV therapeutics, it is the overarching goal of this proposal to apply a rigorous drug development approach to the problem and ultimately identify an anti-RSV therapeutic candidate and at least one mechanistically distinct alternative compound that are suitable for IND-enabling formal development. Narrowly focused drug discovery campaigns are at high risk of early stage failure. We hypothesize that a comprehensive anti-RSV approach interrogating the full host-pathogen interactome for pathogen-directed and host-directed drug candidates has the highest prospect of ultimately yielding viable clinical candidates. This is based on the realization that traditional pathogen-directed therapeutics enjoy an excellent clinical record but can be compromised by emerging viral resistance and/or a narrow pathogen indication spectrum, whereas host-directed antivirals promise to overcome these limitations, yet are at present predominantly still in preclinical development. Building on our multiple-year expertise in the development of myxovirus inhibitors, we have in pilot studies engineered a first in class recombinant RSV expressing a luciferase reporter and developed an innovative drug screening protocol that allows the simultaneous identification of pathogen- directed and host-directed antivirals in a single-well setting. Proof-o-concept implementation of this screen has yielded, amongst others, a novel pathogen-specific small-molecule RSV entry inhibitor with exquisite toxicity profile and an innovative host-directed agonist class of cellular antiviral defense pathways. Lead compounds of both series show nanomolar inhibitory activity against pathogenic RSV variants. To maximize the prospect of success of this program and meet the clinical demand for effective RSV therapy, we will diversify the portfolio of anti-RSV candidates by implementing a full-scale drug screening campaign based on the newly established protocol (aim 1). In parallel, the existing leads and, as discovery advances, novel anti-RSV candidates will be mechanistically characterized and their molecular targets identified (aim 2). Candidates with high developmental potential will be subjected to hit-to-lead synthetic optimization guided by in vitro potency, ADME parameters and small-animal pharmacokinetics profiles (aim 3). The efficacy of lead candidates in alleviating key features of RSV bronchiolitis, airway damage, high lung viral load, mucus expression, and pulmonary obstruction, will be assessed in a newly established clinically relevant mouse model of RSV infection (aim 4).